the better approach

Demineralized Bone Matrix:Development of a Process for Optimal Performance

Introduction

MTF is a non-profit organization founded in 1987 by academic orthopaedic

surgeons dedicated to providing tissue of the highest quality and safety

for transplantation. Everything we do at MTF begins with safety. MTF has

distributed almost 4.2 million allografts since our inception, and we have

never experienced a case of viral disease transmission. MTF’s exemplary

safety record is directly attributed to our commitment to the donor families

and to the tissue recipients we serve. This tremendous commitment

provides our customers with the assurance that this gift of human tissue is

safe and that it comes from a trustworthy source.

We also think beyond safety. While safety governs every decision we make, we

know that quality and efficacy also matter. Current techniques used by some

tissue banks to clean, process and sterilize demineralized allografts have

been shown to be detrimental to the quality of the tissue. These methods vary

widely from bank to bank, because the industry donor criteria and processing

standards are open to interpretation. Demineralized allograft tissue of less-

than-optimal quality may yield a graft that does not perform its intended

function, leading to a less-than-optimal clinical outcome.

Principles of Bone Healing and Graft Incorporation1

Four components are necessary for bone healing and/or bone graft

incorporation: the presence of host cells, a signal to trigger differentiation

of the host cells to bone forming cells, a scaffold or matrix on which the

new bone can form, and an adequate blood supply.

When bone fracture or injury occurs, there is loss of mechanical integrity

of the bone and disruption to the blood supply. The healing cascade begins

immediately. The three phases are: inflammation, repair and remodeling.

Inflammation is the process by which host cells remove debris from the

injured site, prepare the local matrix into a site which can support cell

growth, and enable new bone to be formed. Revascularization, which is

required for new bone to grow, begins in the inflammation phase. Repair

includes the recruitment and differentiation of host cells into osteoblasts,

which in turn produce new bone at the injured site. Lastly, remodeling is

the resorption of immature or extraneous bone coupled with reorientation

of bone along the direction of mechanical loading to provide adequate

structural support. These phases (Figure 1) are regulated by the release of

local cytokines.

Figure 1: Example of the healing cascade in fracture repair. The three phases of fracture repair include A) the inflammatory phase, B) the reparative phase, and C) the remodeling phase.1

Monocytes Mesenchymal cellsMacrophages

RecruitmentPDGF NeovascularizationTGF-ß etc...

FibroblastsOsteoblastsChondrocytes

Cartilage

A

B

C

Intramembranousossification

Osteoclast/osteoblast remodeling occurs on the surface and via osteons

New bone formation and bone healing are influenced by several factors in

the bone graft material, some of which can be controlled, such as:

• Bone forming potential • Porosity • pH

Host factors are not as easily controlled:

• Age • Systemic disease • Vascularity • Presence of infection • Quantity and quality of host cells • Use of anti-inflammatory drugs

Bone grafts are often used as bone void fillers to assist with bone healing.

Grafts can provide support or cell signals. During the healing process,

the bone grafts are incorporated into the host bone by remodeling and/or

creeping substitution. Bone graft materials used as bone void fillers can be

described as osteogenic, osteoinductive and/or osteoconductive.

Osteogenic tissues are capable of forming new bone from living cells.

Osteoprogenitor cells proliferate and differentiate into osteoblasts (bone-

building cells) and eventually into osteocytes (mature bone cells). These

cells represent the osteogenic potential of the graft.1

Osteoinductive tissues are ones which promote chemotaxis, mitogenesis

and formation of osteoprogenitor cells that have osteogenic capacity (as

described above).2 Osteoinductive materials will form bone when implanted

into tissues which would not otherwise form new bone.3

Osteoconductive tissues allow for fibrovascular tissue development and

osteoprogenitor cell invasion of a porous structure. This material then acts

as a temporary scaffold which will be replaced with newly formed bone.2

Rationale for Clinical Use of DBM

Autograft bone provides all three components necessary for bone healing. It

has been widely used during bone grafting procedures due to availability of

donor graft sites and good incorporation upon transplantation. However, the

use of autograft bone requires a second surgical site (to procure bone) which

has associated morbidity risks. Allogeneic demineralized bone matrix (DBM)

eliminates the need for a second surgical site, and its demineralized state

results in bone that is osteoconductive and has osteoinductive potential.

How DBM Works

The exact mechanism of the osteoinductive potential of DBM has not been

very well defined. However, it is thought that the removal of the mineral

component of bone exposes the active bone morphogenetic proteins (BMPs)

present in the DBM while retaining the inherent osteoconductive properties

of the bone. When implanted, the active BMPs are thought to signal the host

mesenchymal cells thus causing the cells to proliferate and differentiate into

chondroblasts, which in turn will form a cartilage matrix. Ultimately, the car-

tilage matrix is converted into a calcified extracellular matrix. This calcified

matrix will become vascularized, and osteoprogenitor cells will form new

bone on this matrix. This will be followed by the formation of bone marrow

and marrow elements.3

Figure 2: Schematic showing differentiation of a stem cell into an active osteoblast, and demonstrating the influence of growth factors.1

What Constitutes Good DBM

MTF has developed and validated a demineralization procedure for

processing bone into DBM. This procedure provides safe, high-quality

allograft DBM and was developed through rigorous testing to ensure that

the osteoinductive potential of the tissue was not compromised.

Many factors contribute to the high quality of MTF DBM, including:

• Quality tissue • Careful processing • A good carrier • Quality control • Scientific evidence

Fibroblast

Chondrocyte

MuscleAdipocyte

BMP’s

IGF’sTGF-ßBMP’s

IGF’sTGF-ßBMP’s

MESENCHYMALSTEM CELL

COMMITEDOSTEOPROGENITOR

CELL

OSTEOBLAST

Quality tissue

MTF’s quality and safety standards consistently meet or exceed the

requirements of the American Association of Tissue Banks (AATB) as well

as the guidelines for screening and testing of tissue donors set forth by the

Food and Drug Administration (FDA). The AATB and FDA set only minimal

guidelines to ensure safety of tissue. Potential MTF donors must pass

through an extensive quality assurance process.

Screening begins at the site of recovery with a comprehensive medical and

social history that includes the cause of death. Tissue and blood samples are

tested for infectious diseases, including hepatitis, HIV and syphilis. A team of

medical/technical specialists from the infectious disease and tissue banking

fields evaluates all information, including test results, before the donor is

released for processing. Figure 3 lists those areas in which MTF voluntarily

defers donors for safety or quality reasons even when not required by the

FDA or AATB.

Careful Processing

To maintain biological integrity, MTF processes all tissue using aseptic

techniques in ISO Class 4 (certified) clean rooms. MTF’s use of these

clean rooms is designed to prevent any environmental contamination

of the tissue and thus eliminates the need for terminal sterilization by

gamma radiation, which has been shown to compromise the biological and

biomechanical integrity of allograft tissue (Figure 4) (see next page).4,5

160 ———––––––––—------———————————————————

140 ———––––––––—------———————————————————

120 ———––––––––—------———————————————————

100 ———––––––––—------———————————————————

80 ———––––––––—------———————————————————

60 ———––––––––—------———————————————————

40 ———––––––––—------———————————————————

20 ———––––––––—------———————————————————

0 ———––––––––————————------———————————— 1 2 3 DBM lot

n 0 kGyn 12 kGyn 18 kGYn 25 kGY

ALP

mm

ol/m

g pr

otei

n/m

in

Figure 4: Gamma radiation decreases osteoinductive potential of DBM powder in a dose-dependent manner, as measured using

an in vitro alkaline phosphatase (ALP) assay. 5

For example, in an experimental study where DBX® Putty was exposed

to a terminal gamma radiation dose of 17.2 kGy, the osteoinductive

potential was reduced by approximately 50% (Figure 5).6

2.5 ———––––––––—------———————————————————

2 ———––––––––—------———————————————————

1.5 ———––––––––—------———————————————————

1 ———––––––––—------———————————————————

0.5 ———––––––––—------———————————————————

0 ———––––––––————————------———————————— Donor 1 Donor 2 Donor 3

n 0 kGyn 17 kGy

Oste

oind

uctiv

ity S

core

Figure 5: Gamma radiation decreases the osteoinductive potential of DBX Putty using the in vivo athymic mouse assay. 6

A Good Carrier

DBM in its native form is a fine powder and as such is difficult to

deliver to an operative site. DBM formulations should be designed

to resist movement under irrigation while maintaining the physical

integrity of the tissue form. Also, ideal DBM formulations should be

provided ready for use and not require any mixing or thawing prior

to use. Therefore, MTF adds a carrier material to the demineralized

bone to improve cohesion and provide an implant with good handling

characteristics.

The carrier used in DBX is sodium hyaluronate which has been proven

to be extremely safe. MTF uses high-quality, medical grade sodium

hyaluronate, ISO 13485 certified, produced through fermentation

processes using Good Manufacturing Practice (GMP) guidelines. It is

designed to be isotonic and non-hemolytic. The sodium hyaluronate

used in DBX tissue is not of animal origin.

Sodium hyaluronate was chosen because it is a polysaccharide

formed by plasma membrane proteins. It is naturally occurring in

the human body in the joints, eyes, extracellular matrix of skin and

musculoskeletal tissue. Sodium hyaluronate plays an essential role in

cell proliferation, migration and adhesion and has been correlated to

angiogenesis.7,8 It also confers positional stability of the tissue.9

Formulations

DBX is a demineralized bone matrix that has osteoinductive potential and is

osteoconductive. It is composed of demineralized bone from human donors

in a biocompatible carrier. The various DBX forms have been designed to

meet surgical needs while maximizing the amount of bone delivered to the

surgical site.

The demineralized bone powder is produced by first milling cortical bone

to the appropriate size (212 – 850µm), followed by removal of the minerals

from the cortical bone via an acid extraction procedure. The demineralized

milled cortical bone is then rinsed and buffered to ensure neutral pH.

Lastly, the demineralized bone is lyophilized (residual moisture content

< 6%) to ensure stability during staging prior to mixing with appropriate

carriers.

DBX Demineralized Bone Matrix is non-hemolytic, ensuring compatibility

with the surrounding autogenous blood cells. The DBX formulations have

been specifically designed to model the pH of human blood.

DBX Paste

DBX Paste provides a flowable consistency of granulated cortical bone in

sodium hyaluronate. The bone content of DBX Paste is 26% bone by weight,

and 78% bone by volume.

DBX Putty

DBX Putty provides a moldable consistency of granulated cortical bone in

sodium hyaluronate. The bone content of DBX Putty is 31% bone by weight,

and 93% bone by volume.

DBX Inject

The DBX Inject package contains a glass syringe preloaded with DBX

Putty and a separate plastic delivery syringe. A compatible cannula and

tamp system is available to accommodate a variety of minimally invasive

techniques. The bone content of DBX Putty is 31% bone by weight, and 93%

bone by volume.

DBX Mix

DBX Mix provides a morselized cortical-cancellous bone texture in sodium

hyaluronate, which eliminates the need to combine bone chips with a DBM.

The bone content of DBX Mix is 35% by weight.

DBX Pre-Formed Shapes

Certain carriers allow MTF to pre-shape the DBM tissue form into

predetermined sizes and shapes. For example, DBX Strip provides a

cohesive and flexible preformed DBM combined with sodium hyaluronate

and gelatin. The bone content of DBX Strip is 45% by weight.

DBX Strip differs slightly from other DBX forms since, in addition to sodium

hyaluronate, its carrier includes porcine gelatin to give it unique handling

characteristics. The combination of demineralized bone, gelatin, and

sodium hyaluronate results in a pre-formed strip formulation.

Quality Control

Every lot of DBX undergoes strict release criteria testing. Sterility is tested

per USP <71>. Each lot is also tested via an in vitro or in vivo assay for

osteoinductive potential.

DBM Processing

MTF’s demineralized bone matrices are produced from both cancellous

and cortical bone which are subjected to controlled cleaning processes

(including hydrogen peroxide and ethanol), followed by demineralization

with hydrochloric acid. MTF’s aseptic processes have been designed to

preserve the biological integrity of the tissue. Each donor lot of DBM is

verified for osteoinductive potential prior to distribution.

Osteoinductivity Testing

Osteoinductive materials have the ability to induce new bone formation

even when implanted into non-bony tissue such as muscle. When bone

is formed in this manner, the material is said to have osteoinductive

potential. Osteoinductive potential can be measured using either in vivo or

in vitro test methods.

MTF has validated three methods to measure DBM osteoinductive

potential prior to distribution: 1) athymic mouse muscle pouch assay, 2)

alkaline phosphatase assay, or 3) BMP-2 content assay. Currently only the

athymic mouse muscle pouch or the alkaline phosphatase assay is used

as lot release method for DBX. Consult the package insert for the specific

method used for each formulation

Athymic Mouse Assay

The athymic mouse assay for in vivo osteoinductive potential is based

upon the Urist10 model and is designed to histologically confirm that DBX

tissues have osteoinductive potential prior to distribution. In this model,

implantation of demineralized bone in the hamstring muscle pouch of

an athymic mouse will result in ectopic bone formation if the bone has

osteoinductive potential. Athymic mice are used as they lack a thymus

gland, and therefore are unable to mount an immune response against the

human tissue. New bone formation is measured histologically after

28 days implantation (minimum) and scored using the scale in Table 1. This

score is expressed as percent of new bone formation in the visualized area.

Figure 6 displays a representative histological section used for scoring

osteoinductive potential.

Table 1: Osteoinductive Potential Scoring Scale OI Score New Bone Formation 0 No evidence of new bone formation 1 1-25% of the section is covered by new bone 2 26-50% of the section is covered by new bone 3 51-75% of the section is covered by new bone 4 >75% of the section is covered by new bone

Figure 6: Example of histological section used to grade osteoinductivity in the athymic mouse Bar = 100 micron.

Alkaline Phosphatase Assay

Alkaline phosphatase (ALP) is an enzyme which is expressed by bone

growing cells during new bone formation. Demineralized bone will

activate these bone growing cells, thus resulting in increased levels of the

ALP enzyme. In vitro test methods have been developed whereby bone

growing cells are exposed to DBM and the resulting level of in vitro ALP

enzyme is then measured. Correlations between the ALP levels and in

vivo osteoinductivity scores from the athymic mouse model demonstrate

that the ALP assay can be used as another means of measuring the

osteoinductive potential of DBM tissue forms.11

BMP-2 Content Assay

In vitro measurement via Enzyme-Linked ImmunoSorbent Assay (ELISA)

of BMP-2 levels in DBM can be used as a surrogate measurement

for the in vivo osteoinductive potential of DBX. Bone induction is a

sequential, multi-step cascade which involves various growth factors.

Bone morphogenetic proteins (and other intrinsic growth factors) in bone

are exposed by the demineralization process. However, BMP-2 has been

shown to be the best single predictor of osteoinductive potential based

on statistical analysis of the correlation between in vitro levels of various

growth factors and in vivo osteoinductivity.12,13

There is a strong correlation (R2 = 0.72) between in vitro ELISA results

on DBM and in vivo osteoinductive potential of DBX Putty in the athymic

mouse model.

Correlation of BMP-2 levels with in vivo 0I(31% DBM in Hy carrier, n=8)

In v

ivo

0I (0

-4 s

core

s)

BMP-2 (pg/g DBM)

4

3

2

1

0 0 5000 10000 15000 20000

y=0.0002x - 0.1308R2 = 0.7167

Figure 7: Correlation of BMP-2 levels in DBM powder and in vivo osteoinductive scores in DBX Putty (R2 = .72)

from the same donor lots (N = 8).

BMP-2 quantified in the final formulation of DBX Putty was found to be of

equivalent levels as in the respective DBM powder (from the same donor

lots). The good correlation (R2 = 0.82) between BMP-2 levels in DBX Putty

and DBM powder (n=18, Figure 8), demonstrates that the addition of sodium

hyaluronate to DBM powder does not alter intrinsic growth factor levels.14

y=-0.7909 x + 2391.6R2=0.8201

BM

P-2

in D

BM

Put

ty (p

g/g

DB

M)

30000

25000

20000

15000

10000

5000

500000

10000 15000 20000 25000 30000

BMP-2 DBM vs DBX Putty (n=18)

BMP-2 in DBM powder (pg/g DBM)

Figure 8: Correlation of BMP-2 levels in DBM powder and DBX Putty (R2 = 0.82) from the same lots (n=18 donor lots).

Indications

DBX is intended for use in voids or gaps that are not intrinsic to the

stability of the bony structure. It is intended for treatment of surgically or

traumatically created osseous defects of the posterolateral spine, pelvis,

and extremities.

Additionally, DBX Putty and Paste are intended for the augmentation of

deficient maxillary and mandibular alveolar ridges and the treatment of

oral/maxillofacial and dental intra-osseous defects.

Please see the package insert for a complete description of indications,

contraindications, and precautions. DBX is for single patient use only.

Expanded Indications for DBX Putty

DBX Putty can be used as an extender in the spine, pelvis, and extremities

with autograft or allograft.

Summary

At MTF, we are driven by our strong commitment to safety. It is because

of this commitment that we continue to maintain an exemplary safety

record, providing our customers with allograft tissue from a source they

can trust. As part of our philosophy, we believe that providing safe tissue

is not enough—we also must not compromise the biological properties

of the demineralized bone. MTF’s processes for bone demineralization

and creation of DBM tissue forms have been designed and validated

to ensure the safety of the allografts without adversely affecting their

biological performance. The processes and studies described here

demonstrate that MTF’s demineralization processes and use of sodium

hyaluronate in DBX have no harmful effects on the natural biological

properties or in vivo performance of the allografts.

The demineralization process and additionally the use of sodium hyaluronate in DBX by MTF yields a safe, effective allograft designed and validated to maintain the natural healing function wof allograft bone.

125 May Street n Edison, NJ 08837 n 800-946-9008 n mtf.org MTF Customer Service: 800-433-6576 © Musculoskeletal Transplant Foundation

BellaDerm® is a registered trademark of MTF© Mentor Worldwide LLC 2013 1307054

References1. Mehta S, Collings C. Orthobiologics: Improving Fracture Care Through Science: Lippincott Williams & Wilkins; 2007.

2. Mouch CS, Einhorn TA. Bone Morphogenetic Proteins and Other Growth Factors to Enhance Fracture Healing and Treatment of Nonunions. In: Lieberman JR, Friedlander GE, editors. Bone Regeneration and Repair: Biology and Clinical Applications: Humana Press; 2005. p 169-194.

3. Boyan BB, McMillan J, Lohman CH, Ranly DM, Schwartz Z. Bone Graft Substitutes: Basic Information for Successful Clinical Use with Special Focus on Synthetic Graft Substitutes. In: Laurencin CT, editor. Bone Graft Substitutes: ASTM; 2003. p 231-259.

4. Vangsness CT, Jr., Wagner PP, Moore TM, Roberts MR. Overview of Safety Issues Concerning the Preparation and Processing of Soft-Tissue Allografts. Arthroscopy 2006; 22(12):1351-8.

5. Han B, Yang Z, Nimni M. Effects of Gamma Irradiation on Osteoinduction Associated with Demineralized Bone Matrix. J Orthop Res 2008; 26(1):75-82.

6. Gertzman AA, Sunwoo M, Raushi D, Dunn M. The Effect of Cold Gamma Radiation Sterilisation on the Properties of Demineralised Bone Matrix. In: Kennedy JF, Philips GO, Williams PA, editors. Sterilisation of tissues using ionising radiations: CRC Press; 2005. p 151-156.

7. Fraser JR, Laurent TC. Turnover and Metabolism in Hyaluronan. In: Evered D, Whelan J, editors. Biology of Hyaluronan: Wiley; 1989. p S41-S59.

8. Orlidge A, D’Amore P. Cell Specific Effects of Glycosaminoglycans on the Attachment and Proliferation of Vascular Wall Components. Microvasc Res 1986;31:S41-S43.

9. Gertzman A, Sunwoo M. A Pilot Study Evaluating Sodium Hyaluronate as a Carrier for Freeze-Dried Demineralized Bone Powder. Cell Tissue Bank 2001;2:S87-S94.

10. Urist MR. Bone: Formation by Autoinduction. Science 1965; 150(698):893-9.

11. Han B, Tang B, Nimni ME. Quantitative and Sensitive In Vitro Assay for Osteoinductive Activity of Demineralized Bone Matrix. J Orthop Res 2003; 21(4):648-54.

12. Blum B, Moseley J, Miller L, Richelsoph K, Haggard W. Measurement of Bone Morphogenetic Proteins and Other Growth Factors in Demineralized Bone Matrix. Orthopedics 2004;27(1 Suppl):s161-5.

13. Murray SS, Brochmann EJ, Harker JO, King E, Lollis RJ, Khaliq SA. A Statistical Model to Allow the Phasing out of the Animal Testing of Demineralised Bone Matrix Products. Altern Lab Anim 2007; 35(4):405-9.

14. Chnari E, Javoroncov M, Gertzman AA, Sunwoo MH, Dunn MG. Bone Morphogenetic Protein 2 (BMP-2) Levels are Predictive of the Osteoinductive Potential of Demineralized Bone Matrix. ORS, 2010.35: 485.